There are cogent data indicating a protective effect of n-3 polyunsaturated fatty acids (PUFA) e.g., eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on colon cancer. In contrast, dietary lipids rich in n-6 PUFA, e.g., linoleic acid (LA) and arachidonic acid (AA), enhance the development of colon tumors. This is significant because the typical Western diet contains 10 to 20 times more n-6 than n-3 PUFA. Unfortunately, to date, a unifying mechanistic hypothesis addressing why n-3 PUFA selectively suppress colon cancer compared to n-6 PUFA (the major dietary form of PUFA in the U.S. diet) is lacking. In the current funding period, we demonstrated that (i) dietary n-3 PUFA induce perturbations in colonocyte membrane microdomain (caveolae) lipid composition, suppressing oncogenic signaling and Ras protein trafficking; (ii) n-3 PUFA supplant arachidonic acid (AA) in colonocyte membrane phospholipids, with EPA in particular being metabolized into a novel 3-series E-prostaglandin (PGE3), a putative antitumorigenic-cyclooxygenase (COX) metabolite; and (iii) the proapoptotic- chemoprotective effect of n-3 PUFA is enhanced when a highly fermentable fiber, pectin (or its fermentation product-butyrate) is added to the diet. We postulate that the failure to address an interaction between dietary fat and fiber may explain why the chemoprotective effects of fiber are obscured in prospective cohort studies. Therefore, the overall goal of this proposal is to further elucidate how dietary n-3 PUFA and fermentable fiber up-regulate apoptosis effector mechanisms in colonocytes, thereby reducing colon cancer risk. An array of experimental models will be used, including in vivo (azoxymethane-injected oxidatively stressed SOD2+/-, Gpx4+/- knock outs, Gpx4Tg and fat-1 transgenic mouse models) and in vitro (normal and malignant transformed mouse and human colonocyte cell lines) in order to test our primary hypothesis that n-3 PUFA and butyrate work in a coordinated manner to potentiate mitochondrial-mediated apoptosis. As a complementary hypothesis, we propose that DHA and possibly EPA alter colonocyte membrane lipid microdomain composition, thereby favorably modulating eicosanoid metabolism and the relay of extracellular signals from surface receptors to downstream signaling networks. The following specific aims are proposed: Aim #1 will determine the mechanisms by which n-3 PUFA and butyrate interaction modulate intrinsic (mitochondria-mediated) cell death signaling in the colon; Aim #2 will determine the mechanisms by which n-3 PUFA alter the spatio-temporal segregation of Ras-dependent signals; and Aim #3 will characterize the putative chemoprotective properties of a novel EPA-derived cyclooxygenase product, PGE3. The consumption of EPA and DHA may prove an effective adjuvant therapy in colon cancer. Therefore, it is both appropriate and timely to determine precisely how n-3 PUFA modulate cell signaling networks and reduce colon cancer risk.A growing body of literature supports the contention that bioactive food components containing n-3 polyunsaturated fatty acids (PUFA) are important in suppressing colon cancer. Consistent with the objectives and scope of PA-07-100, Prioritizing molecular targets for cancer prevention with nutritional combinations , the overall goal of this proposal is to elucidate how dietary n-3 PUFA and fermentable fiber up-regulate apoptosis effector mechanisms in colonocytes, thereby reducing colon cancer risk.